The invention relates to antitumor antibiotics. More particularly, the invention relates to analogs of CC-1065 and the duocarmycins having antitumor antibiotic activity.
(+)-CC-1065 (1) and the duocarmycins 2-3 represent the initial members of a class of exceptionally potent antitumor antibiotics. Members of this class of antitumor antibiotic derive their biological effects through the reversible, stereoelectronically-controlled sequence selective alkylation of duplex DNA. (H. Sugiyama, et al., Tetrahedron Lett. 1990, 31, 7197; C. H. Lin, et al., J. Am. Chem. Soc. 1992, 114, 10658; H. Sugiyama, et al., Tetrahedron Lett. 1993, 34, 2179; K. Yamamoto, et al., Biochemistry 1993, 32, 1059; A. Asai, et al., J. Am. Chem. Soc. 1994, 116, 4171; and D. L. Boger, et al., Tetrahedron 1991, 47, 2661.) (+)-CC-1065 (1) was first disclosed in 1981 by L. J. Hanka, et al.. (J. Am. Chem. Soc. 1981, 103, 7629.) The duocarmycins 2-3 were first disclosed in 1988 and 1990. (Takahashi, et al.. J. Antibiot. 1988, 41, 1915; T. Yasuzawa, et al., Chem. Pharm. Bull. 1988, 36, 3728; M. Ichimura, et al., J. Antibiot. 1988, 41, 1285; M. Ichimura, et al., J. Antibiot. 1990, 43, 1037; M. H. Ichimura, et al., J. Antibiot. 1991, 44, 1045; K. Ohba, et al., J. Antibiot. 1988, 41, 1515; and S. Ishii, J. Antibiot. 1989, 42, 1713.)
Subsequent to their disclosure, extensive efforts have been devoted to establish their duplex DNA alkylation selectivity and its structural origin. (D. L. Boger, Acc. Chem. Res. 1995, 28, 20; D. L. Boger, Proc. Nal. Sci. U.S.A. in press; D. L. Boger, Chemtracts: Org. Chem. 1991, 4, 329; D. L. Boger, In Proceed R. A. Welch Found. Conf. on Chem. Res., XXXV. Chem. at the Frontiers of Medicine 1991, 35, 137; D. L. Boger, In Advances in Heterocyclic Natural Products Synthesis, Vol. 2, Pearson, W. H. Ed.; JAI Press: Greenwich, Conn., 1992, 1-188; D. L. Boger, Pure Appl. Chem. 1993, 65, 1123; D. L. Boger, Pure Appl. Chem. 1994, 66, 837; R. S. Coleman, In Studies in Nat. Prod. Chem., Vol 3, Rahman, A.-u.-, Ed.; Elsevier: Amsterdam, 1989, 301; and D. L. Boger, In Heterocycles in Bioorganic Chemistry; J. Bergman, H. C. van der Plas, and M. Simonyl, Eds; Royal Society of Chemistry: Cambridge, 1991, 103.) Progress has also been made with respect to characterizing the link between DNA alkylation and the ensuing biological properties. (D. L. Boger, et al., Bioorg. Med. Chem. Lett. 1994, 4, 631.) Extensive efforts have also been devoted to define the fundamental principles underlying the relationships between structure, chemical reactivity, and biological properties. (W. Wierenga, et al., Adv. Enzyme Regul. 1986, 25, 141; M. A. Warpehoski, et al., J. Med. Chem. 1988, 31, 590; D. L. Boger, et al., J. Am. Chem. Soc. 1993, 115, 9025; D. L. Boger, et al., J. Am. Chem. Soc. 1992, 114, 10056; H. Muratake, et al., Tetrahedron Lett. 1994, 35, 2573; Y. Fukuda, et al., Tetrahedron 1994, 50, 2793; Y. Fukuda, et al., Tetrahedron 1994, 50, 2809; Y. Fukuda, et al., Bioorg. Med. Chem. Lett. 1992, 2, 755; Y. Fukuda, et al., Tetrahedron Lett. 1990, 31, 6699; W. Wierenga, J. Am. Chem. Soc. 1981, 103, 5621; P. Magnus, et al., J. Am. Chem. Soc. 1987, 109, 2706; G. A. Kraus, et al., J. Org. Chem. 1985, 50, 283; D. L. Boger, et al., J. Am. Chem. Soc. 1988, 110, 1321, 4796; R. E. Bolton, et al., J. Chem. Soc., Perkin Trans. 1 1988, 2491; R. J. Sundberg, et al., J. Org. Chem. 1988, 53, 5097; R. J. Sundberg, et al., J. Org. Chem. 1991, 56, 3048; V. P. Martin, Helv. Chim. Acta 1989, 72, 1554; M. Toyota, et al., J. Chem. Soc., Perkin Trans. 1 1992, 547; and L. F. Tietze, et al., J. Org. Chem. 1994, 59, 192.) The relationships between structure, chemical reactivity, and biological properties of CI-based analogs have also been characterized. (D. L. Boger, et al., Proc. Natl. Acad. Sci. U.S.A. 1991, 88, 1431; D. L. Boger, et al., J. Am. Chem. Soc. 1991, 113, 3980; D. L. Boger, et al., J. Org. Chem. 1989, 54, 1238; D. L. Boger, et al., J. Am. Chem. Soc. 1990, 112, 5230; K. J. Drost, et al., J. Org. Chem. 1989, 54, 5985; J. H. Tidwell, et al., J. Org. Chem. 1992, 57, 6380; J. Sundberg, et al., Tetrahedron Lett. 1986, 27, 2687; Y. Wang, et al., Heterocycles 1993, 36, 1399; Y. Wang, et al., J. Med. Chem. 1993, 36, 4172; L. F. Tietze, et al., Chem. Ber. 1993, 126, 2733; and T. Sakamoto, et al., J. Chem. Soc., Perkin Trans. 1 1993, 1941.) The relationships between structure, chemical reactivity, and biological properties of C2BI-based analogs have also been characterized. (D. L. Boger, et al., J. Am. Chem. Soc. 1992, 114, 9318; and D. L. Boger, et al., Bioorg. Med. Chem. 1993, 1, 27.) The relationships between structure, chemical reactivity, and biological properties of CBQ-based analogs have also been characterized. (D. L. Boger, et al., J. Am. Chem. Soc. 1994, 116, 6461; and D. L. Boger, et al., J. Am. Chem. Soc. 1994, 116, 11335.) F. Mohamadi et al. have characterized the relationships between structure, chemical reactivity, and biological properties of CFI-based analogs (J. Med Chem. 1994, 37, 232.) A p-quinonemethide analog was characterized by D. L. Boger, et al.. (J. Org. Chem. 1994, 59, 4943.)
Concurrent with the above structure/function studies, substantial efforts have been devoted to developing potential clinical candidates based on the natural product structures having enhanced in vivo efficacy. Compounds 4-8 are analogs of the natural product structures having enhanced in vivo efficacy with clinical potential. (D. L. Boger, et al., J. Org. Chem. 1984, 49, 2240; M. A. Warephoski, M. A. Tetrahedron Lett. 1986, 27, 4103; Li, L. H.; Invest New Drugs 1991, 9, 137; B. K. Bhuyan, et al., Cancer Res. 1992, 52, 5687; B. K. Bhuyan, et al., Cancer Res. 1993, 53, 1354; L. H. Li, et al., Cancer Res. 1992, 52, 4904; M. A. Mitchell, et al., J. Am. Chem. Soc. 1991, 113, 8994. Lee, C.-S.; Gibson, N. W. Cancer Res. 1991, 51, 6586. Lee, C.-S.; Gibson, N. W. Biochemistry 1993, 32, 9108; Wierenga, W. Drugs Fut. 1991, 16, 741; K. Gomi, et al., Jpn. J. Cancer Res. 1992, 83, 113. Okamoto, A.; Okabe, M.; Gomi, K. Jpn. J. Cancer Res. 1993, 84, 93; E. Kobayashi, et al., Cancer Res. 1994, 54, 2404; and H. Ogasawara, Jpn. J. Cancer Res. 1994, 85, 418. ) A Phase I clinical trial one one drug candidate in this class is described by G. F. Fleming, et al., (J. Natl. Cancer Inst. 1994, 86, 368.) Efforts have also focused on the development of analogs having decreased delayed toxicity as compared to the natural form of (+)-CC-1065. (J. P. McGovren, et al., Cancer Res. 1993, 53, 5690.) Importantly, this unusual property has not been observed with ent-(xe2x88x92)-CC-1065, although it is equally cytotoxic, and is not observed with the naturally-derived duocarmycins as well as simplified analogs of the natural products.
The first preparation and examination of agents containing the 1,2,9,9a-tetrahydrocyclopropa[c]benz[e]indol-4-one (CBI) alkylation subunit were described in connection with efforts to evaluate CC-1065 and duocarmycin analogs bearing deep-seated structural alterations in the alkylation subunit. (D. L. Boger, et al., J. Am. Chem. Soc. 1989, 111, 6461; and D. L. Boger, et al., J. Org. Chem. 1990, 55, 5823.) These agents were employed as tools to identify the structural features of compounds 1-3 associated with their sequence selective alkylation of duplex DNA and to define the fundamental relationships between structure, chemical or functional reactivity and biological properties.
Prior to the present invention, it had been assumed that the unique alkylating activity of the naturally occurring CPI subunit of CC-1065 would be degraded if this portion of the molecule were structurally altered. (L. H. Hurley, et al., Science 1984, 226, 843; V. L. Reynolds, et al., Biochemistry 1985, 24, 6228. L. H. Hurley, et al., Biochemistry 1988, 27, 3886; L. H. Hurley, et al., J. Am. Chem. Soc. 1990, 112, 4633; M. A. Warpehoski, et al., J. Biochemistry 1992, 31, 2502; D. L. Boger, et al., Bioorg. Med. Chem. 1994, 2, 115; D. L. Boger, et al., J. Am. Chem. Soc. 1990, 112, 4623; M. A. Warpehoski, et al., In Advances in DNA Sequence Specific Agents; Hurley, L. H., Ed.; JAI Press: Greenwich, Conn., 1992, Vol 1, 217; M. A. Warpehoski, Drugs Fut. 1991, 16, 131; M. A. Warpehoski, et al., in Molecular Basis of Specificity in Nucleic Acid-Drug Interactions; B. Pullman and J. Jortner, Eds.; Kluwer: Netherlands; 1990, 531; M. A. Warpehoski, et al., Chem. Res. Toxicol. 1988, 1, 315; Hurley, L. H.;. In Molecular Aspects of Anticancer Drug-DNA Interactions; Neidle, S., Waring, M., Eds.; CRC Press: Ann Arbor, Mich. 1993, Vol 1, 89; and L. H. Hurley, et al., Acc. Chem. Res. 1986, 19, 230.) The above assumption is disclosed herein to be inaccurate. Furthermore, the natural enantiomers of the CBI-based analogs of (+)-CC-1065, have been shown to be approximately four times more stable chemically and approximately four times more potent biologically as compared to the corresponding agents incorporating the natural CPI alkylation subunit of CC-1065. (D. L. Boger, et al., Tetrahedron Lett. 1990, 31, 793; D. L. Boger, et al., J. Org. Chem. 1992, 57, 2873; and D. L. Boger, et al., J. Org. Chem. 1995, 60, 0000.) The CBI analogs are also considerably more synthetically accessible as compared to the naturally occuring CPI compounds. (+)-CBI-indole2 (27) exhibits cytotoxic potency comparable to that of the (+)-CC-1065 and greater (4xc3x97) than that of the potential clinical candidate (+)-CPI-indole2 (4, U71,184) introduced by Upjohn. (+)-CBI-indole2 (27) also exhibits potent and efficacious in vivo antitumor activity. (D. L. Boger, et al., Bioorg. Med. Chem. Lett. 1991, 1, 115.) (+)-CBI-indole2 (27) was the first efficacious antitumor activity by a CC-1065 analog possessing a structurally altered and simplified DNA alkylation subunit. Moreover, the agent further lacked the delayed fatal toxicity characteristic of (+)-CC-1065.
The natural enantiomers of the CBI-based analogs have been shown to alkylate DNA with an unaltered sequence selectivity as compared to the corresponding CPI analog. (D. L. Boger, et al., J. Am. Chem. Soc. 1994, 116, 7996; and P. A. Aristoff, et al., J. Med Chem. 1993, 36, 1956.) Furthermore, the DNA alkylation of CBI-based analogs occurs at an enhanced rate as compared to the corresponding CPI analogs (D. L. Boger, et al., J. Am. Chem. Soc. 1991, 113, 2779) and with a greater efficiency than the corresponding CPI analog. (D. L. Boger, et al., J. Am. Chem. Soc. 1992, 114, 5487)
Refined models of the DNA alkylation reactions of the duocarmycins have been developed which accomodate the reversed and offset AT-rich adenine N3 DNA alkylation selectivity of the enantiomeric agents and their structural analogs. (D. L. Boger, et al.,i J. Org. Chem. 1990, 55, 4499; D. L. Boger, et al., J. Am. Chem. Soc. 1990, 112, 8961; D. L. Boger, et al., J. Am. Chem. Soc. 1991, 113, 6645; D. L. Boger, et al., J. Am. Chem. Soc. 1993, 115, 9872; D. L. Boger, et al., Bioorg. Med. Chem. Lett. 1992, 2, 759; and D. L. Boger, et al., J. Am. Chem. Soc. 1994, 116, 1635.) A similar refined model of the DNA alkylation reactions of CC-1065 have been developed which also accomodate the reversed and offset AT-rich adenine N3 DNA alkylation selectivity of the enantiomeric agents and their structural analogs. (D. L. Boger, et al., Bioorg. Med. Chem. 1994, 2, 115; and D. L. Boger, et al., J. Am. Chem. Soc. 1990, 112, 4623.) These models teach that the diastereomeric adducts derived from the unnatural enantiomers suffer a significant destabilizing steric interaction between the CPI C7 center (CH3) or the CBI C8 center with the base adjacent to the alkylated adenine which is not present with the natural enantiomer adducts. Moreover, the distinguishing features of the natural and unnatural enantiomers diminish or disappear as the inherent steric bulk surrounding this center is reduced or removed. Because of the unnatural enantiomer sensitivity to destabilizing steric interactions surrounding the CPI C7 or CBI C8 center, the unnatural enantiomers of the CBI-based analogs are particularly more effective than the corresponding CPI analog displaying an even more enhanced relative rate and efficiency of DNA alkylation.
An extensive study of analogs of the potent antitumor antibiotics CC-1065 and the duocarmycins which incorporate the 1,2,9,9a-tetrahydrocyclopropa[c]benz[e]indol-4-one (CBI) alkylation subunit are detailed. In contrast to early speculation, deep-seated modifications in the CC-1065 and duocarmycin alkylation subunits are well tolerated and the CBI-based analogs proved to be potent cytotoxic agents and efficacious antitumor compounds. Full details of studies defining a direct relationship between functional stability and in vitro cytotoxic potency are described. As such, the readily accessible CBI-based analogs were found to be 4xc3x97 more stable and 4xc3x97 more potent than the corresponding analogs containing the authentic CPI alkylation subunit of CC-1065 and comparable in potency to agents containing the authentic alkylation subunit of duocarmycin SA. Similarly, the CBI-based agents alkylate DNA with an unaltered sequence selectivity at an enhanced rate and with a greater efficiency than the corresponding CPI analog and were comparable to the corresponding analog incorporating the duocarmycin SA alkylation subunit. Systematic and extensive modifications and simplifications in the DNA binding subunits attached to CBI were explored with the comparisons of both enantiomers of 1-3 with both enantiomers of 18-24, 25-29, 57-61, 62-65, 66-68, 72-73 and 78-79.
Simple Derivatives of CBI. Role of the N2 Substituent and Validation of a Direct Relationship Between Functional Stability and In Vitro Cytotoxic Potency. Substantial quantities of optically active natural-(1S)- and ent-(1R)-15 were prepared through use of our original synthesis of CBI and its precursors, as referenced above with two recent modifications. (D. L. Boger, et al., J. Org. Chem. 1992, 57, 2873; and D. L. Boger, et al., J. Org. Chem. 1995, 60, 0000.) The most efficient approach now proceeds in 9 steps and in 38% overall yield from commercially available 1,3-dihydroxynapthalene based on a key 5-exo-trig aryl radical-alkene cyclization for the direct preparation of N-BOC-5-benzyloxy-1-hydroxymethyl-1,2-dihydro-3H-benz[e]indole. Moreover, the initial resolution we described based on the chromatographic separation of the diastereomeric (R)-O-acetyl madelate esters of the primary alcohol precursor to 15 which has been adopted by others has been since improved in our efforts. The more advanced synthetic intermediate 15, and in fact the penultimate intermediate to the CBI-based analogs, may be directly and more efficiently resolved (xcex1=1.28) on an analytical or preparative Daicel Chiralcel OD column without recourse to diastereomeric derivatization. For our purposes, 20 mg of 15 could be separated in a single injection on a semipreparative 10 xcexcm, 2xc3x9725 cm OD HPLC column (5% i-PrOH-hexane, 8 mL/min) with a 90-100% recovery of the total sample. Conversion of natural (1S)- and ent-(1R)-15 to (+)- and ent-(xe2x88x92)-N-BOC-CBI (9), and (+)- and ent-(xe2x88x92)-CBI (17) have been detailed in our initial studies, and provided our comparison standards for the studies detailed below (FIG. 1).
Initial studies conducted with simple derivatives of the (+)-CC-1065 alkylation subunit (CPI) led to the proposal that there exists a direct relationship between an agent""s reactivity and in vitro cytotoxic potency (L1210, IC50) and established the expectation that the biological potency may be enhanced as their electrophilic reactivity is increased. In our complementary series of studies conducted with agents containing deep-seated modifications in the alkylation subunit including 9-14, the reverse relationship has been observed and the agents possessing the greatest chemical solvolysis stability exhibited the most potent in vitro cytotoxic activity. Moreover, a direct relationship between solvolytic stability and biological potency has been observed and proved to be general with both simple and advanced analogs of the natural products.
As a consequence of these studies, we became interested in the inherent role of the CC-1065 and duocarmycin N2 substituent. Consequently, the simple derivatives 21-24 of (+)-CBI were prepared for examination and, by virtue of their structural similarities, were expected to more accurately reflect a potential relationship between functional reactivity and biological potency than the preceding studies. Treatment of crude, freshly prepared 16 with methyl isocyanate (2 equiv, 3 equiv NaHCO3, THF, 0-25xc2x0 C., 1 h, 83%) provided 18 and attempts to conduct this reaction in more polar solvents including DMF or in the presence of a stronger base (i.e. Et3N) which promotes competitive closure of 16 to CBI (17) led to lower conversions. Spirocyclization of 18 to 21 was effected by treatment with DBU (2 equiv, DMF, 4xc2x0 C., 48 h, 90%) and the use of shorter reaction periods (24 h, 55%) or less polar solvents (THF, 18 h, 35%) provided lower conversions. Treatment of the freshly generated crude indoline hydrochloride salt 16 with ClCO2CH3 (2 equiv, 3 equiv NaHCO3, THF, 0-25xc2x0 C., 1.5 h) provided 19 (100%) in quantitative conversion. Spirocyclization of 19 to provide 22 was effected by treatment with DBU (2 equiv, THF, 0xc2x0 C., 48 h and 25xc2x0 C., 10 h, 93%) and the rate of ring closure of 19 to 22 only became significant at 25xc2x0 C. under these conditions. Even treatment of 19 with K2CO3 (1.5 equiv, THF, 25xc2x0 C., 5 d, 51%) provided 22 albeit with this latter reaction requiring a long reaction period. Similarly, treatment of crude 16 with ClCOCH2CH3 (2 equiv, 3 equiv NaHCO3, THF, 0-25xc2x0 C., 5 h or 0xc2x0 C., 1H) cleanly provided 20 (94-98%). Spirocyclization of 20 to cleanly provide 23 was effected by simply dissolving 20 in a 1:1 mixture of 5% aqueous NaHCO3-THF (25xc2x0 C., 5-10 h, 97%) and stirring the resulting two-phase reaction mixture at room temperature. Given the ease of hydrolysis of N-acyl-CBI derivatives upon exposure to aqueous base, it is of special note that this set of reaction conditions worked so well for 23. Lower conversions to 23 were observed upon treatment of 20 with DBU (2 equiv, THF, 0-25xc2x0 C., 18 h) and, although this was not examined in detail, can be attributed to a slow cyclization under the reaction conditions resulting in significant amounts of recovered, unreacted 20. Surprisingly, the most challenging of the derivatives to prepare was 24. Attempts to couple freshly generated 16 with ClSO2CH2CH3 under a wide range of reaction conditions deliberately generating or avoiding sulfene formation suffered from competitive or preferential O-sulfonylation or competitive closure to 17. Although this approach could be used to generate 24, the most productive preparation was accomplished simply by reaction of the sodium salt of CBI (17, 2.5 equiv NaH, THF, 0xc2x0 C., 10 min) with ClSO2CH2CH3 (7 equiv, 3 equiv Et3N, 25xc2x0 C., 3 h, 45%) to provide 24 directly.
The acid-catalyzed solvolysis of 21-24 conducted at pH 3 (CH3OHxe2x80x94H2O) were followed spectrophotometrically by UV with the disappearance of the characteristic long-wavelength absorption band of the CBI chromophore and with the appearance of a short-wavelength absorption band attributable to the seco-N-BOC-CBI derivative, FIGS. 19A-19B. The results of these studies along with the cytotoxic activities of 21-24 are summarized in FIGS. 20A-20D. The cytotoxic activity of the full set of agents examined and the comparisons with the related CPI-based agents are summarized in FIG. 7.
The comparisons of 21-24 revealed a direct, linear relationship between the cytotoxic potency (L1210, log 1/IC50) and the solvolytic stability (xe2x88x92log ksolv, pH 3) of the agents (FIGS. 20A-20D). Thus, similar to the trend observed with 9-14, the solvolytically more stable derivatives of CBI proved to be the most potent. Similarly, a linear relationship was found between the electron-withdrawing properties of the N2 substituents (Hammett "sgr"p constant) and the solvolysis reactivity (xe2x88x92log ksolv, pH 3) of the agents with the strongest electron-withdrawing substituents providing the most stable agents (FIGS. 20A-20D). This latter relationship reflects the influence of the N2 substituent on the ease of C4 carbonyl protonation required for catalysis of solvolysis and cyclopropyl ring cleavage with the stronger electron-withdrawing N2 substituents exhibiting slower solvolysis rates. Less obvious but more fundamental, the observations were found to follow a predictable linear relationship between the cytotoxic potency (L1210, log 1/IC50) and the electron-withdrawing properties of the N2 substituent (Hammett "sgr"p) with the strongest electron-withdrawing substituents providing the biologically most potent agents (FIGS. 20A-20D).
These fundamental correlations between the electron-withdrawing properties of the N2 substituent, the functional reactivity of the agents, and their biological potency should prove useful in the predictable design of new analogs. In fact, it is this fundamental validation of the direct relationship between functional stability and biological potency that suggests that the CBI-based analogs, which are 4xc3x97 more stable than the corresponding CPI-based analogs, offer rationally-based advantages that may be expected to be even further enhanced by the inherent selectivity that is intrinsic in the diminished reactivity. For agents in this class which possess sufficient reactivity to effectively alkylate duplex DNA, the chemically more stable agents may be expected to constitute the biologically more potent agents. Presumably, this may be attributed to the more effective delivery of the more stable agents to their intracellular target, and the solvolysis rates may be taken to represent a general measure of the relative functional reactivity. Notably, the consumption of the agent in route to its intracellular target need not be simply nonproductive solvolysis but competitive alkylation of nonproductive extra- and intracellular sites as well including the potential of nonproductive sites within duplex DNA. Since the chemically more stable agents provide thermodynamically less stable and more readily reversed addition products, the observations may also represent a more effective thermodynamic partitioning of the agents to their productive intracellular target or site(s).
Consistent with prior observations, the corresponding seco agents 15 and 18-20 which serve as the immediate synthetic precursors to 9 and 21-23 exhibited a cytotoxic potency indistinguishable from that of the corresponding agent incorporating the preformed cyclopropane ring. Since simple C4 phenol O-alkyl (CH3, CH2Ph) and O-acyl derivatives of 15 exhibit substantially diminished cytotoxic potency (10-100xc3x97), this equivalency of the seco precursors 15 and 18-20 with 9 and 21-23 most likely may be attributed to their facile closure to the biologically relevant and more potent cyclopropane containing agents. Notably, such observations have been instrumental in the successful development of prodrug strategies for the advanced analogs of the natural products including 6-8.
Although we have described an extensive account of the DNA alkylation properties of (+)- and ent-(xe2x88x92)-N-BOC-CBI (9) and their comparison with those of (+)- and ent-(xe2x88x92)-N-BOC-CPI (11) the properties of 21-24 and their relationship to the biological evaluations are worth summarizing. The agents 21-24 behaved in a manner comparable to 9. The natural and unnatural enantiomers of 21-24 were substantially less efficient (ca. 104xc3x97), less selective (selectivity=5xe2x80x2-AA greater than 5xe2x80x2-TA) with 40-45% of all adenines alkylated over a 10-fold agent concentration range, and exhibited an altered DNA alkylation profile than (+)- or ent-(xe2x88x92)-1-3. Moreover, the natural enantiomers of 21-24, like (+)- vs ent-(xe2x88x92)-9, proved to be approximately 5-10xc3x97 more efficient than the unnatural enantiomers at alkylating DNA, but were found to exhibit the same selectivity and alkylate the same sites. This alkylation selectivity of 21-24, like that of 9, was identical to that of (+)- or ent-(xe2x88x92)-N-BOC-CPI. However, both the natural enantiomers (5xc3x97) and especially the unnatural enantiomers (10-100xc3x97) of the CBI-based agents were more effective at alkylating DNA than the corresponding CPI-based agent consistent with models that we have discussed in detail. Importantly, the less reactive CBI-based agents were found to alkylate DNA at a faster rate, with a greater efficiency, and with a slightly greater selectivity among the available sites than the corresponding CPI-based agent. This may be interpreted in terms of agents steric accessibility to the adenine N3 alkylation site where the C7 methyl group of the CPI alkylation subunit sterically decelerates the rate of DNA alkylation to the extent that the less reactive, but more accessible, CBI subunit alkylates DNA at a more rapid rate. Since the unnatural enantiomers are even more sensitive to destabilizing steric interactions at the CPI C7 or CBI C8 position, the unnatural enantiomers of the CBI-based agents are particularly more effective than the CPI-based agents.
Advanced Analogs of CC-1065 and the Duocarmycins: Simplification of the DNA Binding Subunits. The preparation and evaluation of both enantiomers of CBI-CDPI2 (25), CBI-CDPI1 (26), CBI-indole2 (27), CBI-indole1 (28), and CBI-TMI (29) and their corresponding seco precursors 30-34 have been disclosed in our early studies and their detailed comparisons with both enantiomers of CC-1065 or the duocarmycins described. More recently, 27, 28, and CBI-PDE-I2 have been disclosed by Aristoff and co-workers. The comparative cytotoxic activity of these prior agents prepared in our studies is summarized in FIG. 9 along with that of the corresponding CPI-based analog.
In an extension of our investigations which first revealed efficacious antitumor activity for 27, we have expanded the studies to the preparation and evaluation of 57-61, a larger series based on 27. The DNA binding subunits of CC-1065 and the duocarmycins contribute in several ways to the properties of the natural products. They contribute significantly to the DNA binding affinity which serves both to increase the rate of DNA alkylation relative to 9 and to thermodynamically stabilize the inherently reversible DNA alkylation reaction. While the former has been suggested to be the origin of the differences in the cytotoxic potency of 1 and 11 by others based principally on the comparisons of (+)-N-BOC-CPI (11), (+)-CPI-indole1, and (+)-CPI-indole2, we have proposed that it is the latter that constitutes the biologically significant distinction. This thermodynamic versus kinetic distinction was first proposed before the reversibility of the DNA alkylation reaction was experimentally verified and was based in part on the observation that the cytotoxic potency of a class of agents would plateau. For example, (+)-CC-1065, (+)-CPI-PDE-I1, and (+)-CPI-CDPIn (n=1-3) were found to be indistinguishable in our cytotoxic assays (IC50=20 pM, L1210). Although the five agents exhibit large differences in their rates of DNA alkylation, all five form thermodynamically stable adducts under physiological conditions. We attribute the increase in cytotoxic potency of CPI-CDPIn (n=1-3) vs 11 to noncovalent binding stabilization of the reversible DNA adduct formation and that it is the simple event not extent of this stabilization that results in their essentially equivalent properties. This interpretation further suggests that CPI-indole, and CBI-indole1 lack the sufficient stabilization for observation of full potency. Moreover, the interpretation is consistent with the observation that a maximum potency is achievable and that the level of this potency is directly related to the functional stability of the agents. Thus, the CBI-based agents examined to date exhibit a similar plateau of potency (5 pM, L1210) but at a level 4xc3x97 more potent than that of the corresponding CPI-based agents (20 pM, L1210).
In addition, the DNA binding subunits of CC-1065 contribute to a strong AT-rich DNA binding selectivity which we have recently shown not only contributes to the alkylation selectivity of the agents but exerts an overriding dominate control. In early studies, we were able to demonstrate that the noncovalent binding affinity was derived nearly exclusively from stabilizing van der Waals contacts and hydrophobic binding. Not only did the studies suggest that CC-1065 is best represented as a selective alkylating agent superimposed on the trimer skeleton but removal of the peripheral methoxy and hydroxy substituents (PDE-I-CDPI) had no effect on its noncovalent AT-rich binding selectivity and little effect on its binding affinity. This dependence on hydrophobic binding stabilization results in preferential binding in the narrower, deeper AT-rich regions of the minor groove where the stabilizing van der Waals contacts are maximal (xcex94Gxc2x0=9.5-11.5 kcal/mol). Moreover, such studies suggested seminal ways in which the DNA binding subunits could be simplified (removal of polar substituents) without altering the characteristics responsible for the essential DNA binding affinity or selectivity.
The DNA binding subunits of the agents may also have a significant impact on the physical properties and characteristics of the agents. Most apparent is the remarkable solubility properties of CC-1065 which is essentially insoluble in all solvents except DMSO or DMF including polar protic or aprotic solvents, water, or nonpolar solvents. A major impact that structural variations in the central and right hand subunits may have is in the solubility properties of the agent and hence its biodistribution and bioavailability.
Finally, we have speculated that the extent of the noncovalent binding stabilization of the inherently reversible DNA alkylation reaction may be responsible for the unusual, delayed toxicity of CC-1065. That is, the extensive noncovalent binding stabilization of 1 that renders its DNA alkylation reaction irreversible while that of simpler agents including 2-3 are slowly reversible under physiological conditions offers a potential explanation for the apparently confusing toxicity profile among the analogs detailed to date. The only agents that have exhibited the delayed toxicity that we are aware of are (+)-CC-1065 (1), (+)-CPI-CDPI2, and (+)-CBI-PDE-I2. Each provide irreversible adduct formation under physiological conditions, and the unnatural enantiomers of each, which form inherently less stable and more reversible adducts, do not exhibit the delayed toxicity. Although speculative, it does suggest that simplified DNA binding subunits which provide sufficient but not extensive binding stabilization of the reversible DNA adduct might offer important advantages that relate to the inherent repair or reversal of nonproductive DNA alkylation sites. Moreover, this would also provide a further strong rationale for the use of less reactive alkylation subunits (CBI versus CPI) whose DNA adducts, while stable, are inherently less stable and more readily reversed.
The preparation of the expanded series of agents 57-61 and their corresponding seco derivatives 52-56 is summarized in FIG. 10. The simplified DNA binding subunits were assembled by coupling methyl 5-aminoindole-2-carboxylate (35) or methyl 5-aminobenzoxazole-2-carboxylate (36) with 37-39. Hydrolysis of the methyl esters 40-45 (LiOH, THFxe2x80x94CH3OHxe2x80x94H2O, 25xc2x0 C.) followed by coupling of the carboxylic acids 46-51 with freshly generated 16 (EDCI, DMF, 25xc2x0 C.) deliberately conducted in the absence of added base provided excellent yields of the seco agents 32 and 52-56. Spirocyclization of 32 and 52-56 was effected by treatment with NaH, DBN, or P4-tBu and provided the agents 27 (CBI-indole2) and 57-61.
The results of the cytotoxic evaluations of the agents are summarized in FIG. 11 along with those of CBI-indole2 (27) and CPI-indole2. Several aspects of these comparative evaluations are notable. First, the natural enantiomers are substantially more potent than the unnatural enantiomers (130-1000xc3x97). In addition, the seco agents 32 and 52-56 exhibited the same levels of cytotoxic activity as the cyclopropane containing agents where compared although this was not investigated in detail. Most notably and with the exception of 60, the cytotoxic potency of natural enantiomers of the new agents were equivalent to or exceeded those of 27 and 57 and all were 2-6xc3x97 more potent than the corresponding CPI analog. Moreover, the potencies of 32 and 52-56 approach or are equivalent with the ceiling of potency observed with 25-36 (5 pM).
Although we have described an extensive account of the DNA alkylation properties of both enantiomers of 25-27, 28, and 29 elsewhere, their comparisons with the corresponding CPI-based agents and their relationship to the biological evaluations merit summarizing. In these studies, a detailed investigation leading to the definition of the 3.5-5 base pair AT-rich adenine N3 alkylation selectivity of the agents were disclosed for both the natural and unnatural enantiomers, models were disclosed which accommodate the reversed binding orientations and offset AT-rich alkylation selectivity, and a beautiful explanation emerged which explains the diminished DNA capabilities of the unnatural enantiomers. Moreover, a clearer picture of the origin of the DNA alkylation selectivity and the structural features of the agents responsible have emerged from these studies. In a detailed comparative examination of the DNA alkylation properties of the CBI-based agents and the corresponding CPI-based analog or duocarmycin SA based agent, they have been found to exhibit identical DNA alkylation selectivities. This is nicely illustrated in FIGS. 3 and 4 with the comparisons of CBI-indole2 (27)/CPI-indole2 (4) and CBI-TMI (29)/duocarmycin SA (2), respectively. In addition, the CBI-based agents have been shown to alkylate DNA both at a faster rate and with a greater efficiency than the corresponding CPI-based agent. This is nicely illustrated in FIG. 21 with the comparison of (+)-CBI-indole2 (27) and (+)-CPI-indole2 (4) where 27 is 10xc3x97 more efficient at alkylating w794 (4xc2x0 C. or 37xc2x0 C., data for latter not shown). Moreover, when the relative rates of DNA alkylation were directly compared at the single high affinity site of w794 DNA, that of CBI-indole2 was considerably faster, k(27)/k(4)=14, FIGS. 23A-23B. In contrast, the natural enantiomer of CBI-based agents and corresponding duocarmycin SA based agents have been found to alkylate w794 DNA with essentially indistinguishable efficiencies (FIG. 22) and at comparable rates, k(29)/k(2)=0.9, FIGS. 23A-23B.
In addition, because of the unnatural enantiomer sensitivity to destabilizing steric interactions surrounding the duocarmycin C7, CPI C7 or CBI C8 center, the unnatural enantiomers of the simpler CBI-based analogs are approximately 4-100xc3x97 less effective than the natural enantiomers. In comparison, the unnatural enantiomers of the CPI-based analogs are 10-1000xc3x97 less effective and the duocarmycin SA based analogs or agents are 1-10xc3x97 less effective in both the cytotoxic assays and in their relative DNA alkylation rate or efficiency. Moreover, this distinction in the enantiomers diminishes only with the larger agents, ie. 25, where the extensive noncovalent binding interactions are sufficiently large to overcome the destabilizing steric interactions of the unnatural enantiomer alkylation. Importantly, these trends follow closely the relative cytotoxic potency of the agents, the relative stabilities of the three classes of agents, and highlight the enhanced distinctions of the CBI- versus CPI-based analogs and the comparable properties of the duocarmycin SA and CBI-based agents. Fundamental to members of this class of antitumor antibiotics, the natural enantiomers of the agents were found to follow a well-defined relationship between solvolysis (functional) stability (xe2x88x92log k, pH 3) and cytotoxic potency (1/log IC50, L1210) where the chemically more stable agents within a given class exert the greatest potency, FIGS. 24A-24E. FIGS. 24A-24E include data for 4-6 available classes of agents that bear five different DNA binding subunits which we have examined, and although this relationship is undoubtedly a second order polynomial indicative of a parabolic relationship that will exhibit an optimal stability-reactivity/potency, the agents employed in FIGS. 24A-24E lie in a near linear range of such a plot. What is unmistakable in the comparisons, is the fundamental direct correlation between functional (solvolytic) stability and cytotoxic potency.
CBI-CDPBO1 and CBI-CDPBI1: Deep-Seated Structural Variations in the DNA Binding Subunits. The efforts of Lown and Dervan have demonstrated that the distamycin AT-rich noncovalent binding selectivity may be altered to accommodate a G-C base-pair or to exhibit progressively altered AT-GC rich binding selectivity through introduction of a nitrogen within the backbone core structure capable of serving as hydrogen bonding acceptor. Accordingly, we have investigated whether similar changes in the core structure of CC-1065 would impact on its DNA binding selectivity and resulting DNA alkylation selectivity. Key to the importance of this examination was the recognition that the more rigid structure of CC-1065, its rigid helical bound conformation, and its near exclusive dependence on stabilizing van der Waals contacts and hydrophobic binding which dictates the preference for binding and alkylation within the narrower, deeper AT-rich minor groove may not be so easily overridden by introduction of a single hydrogen bond acceptor or donor.
In the conduct of these studies, we reported the preparation of (+)- and ent-(xe2x88x92)-CBI-CDPBO1 (62), (+)- and ent-(xe2x88x92)-CBI-CDPBI1 (64) and their corresponding seco precursors 63 and 65 bearing deep-seated modifications in the DNA binding subunit including the incorporation of a nitrogen atom capable of functioning as a hydrogen bond acceptor (CDPBO, CDPBI) or hydrogen bond donor (CDPBI) on their inside convex face which is projected to be in intimate contact with the minor groove floor.
The initial comparisons were made with agents containing a single DNA binding subunit where the single deep-seated structural modification in the DNA binding subunit might be expected to exert a more pronounced effect. In these studies, the DNA alkylation selectivities and efficiencies of the natural enantiomers of 62 and 64 were found to be essentially identical. Moreover, both were approximately 100xc3x97 less efficient at alkylating DNA than (+)-CBI-CDPI1 (26). Thus, the simple incorporation of a single nitrogen into 64 versus 26 has a pronounced and detrimental effect on the relative efficiency of DNA alkylation. Identical to trends detailed in our prior work on the CBI-derived agents, the unnatural enantiomers of 62 abd 64 proved to be 10-100xc3x97 less efficient at alkylating DNA than the corresponding natural enantiomers.
More interesting was the observed DNA alkylation selectivities of 62 and 64. The DNA alkylation selectivities of (+)-62 and (+)-64 were essentially identical and both were comparable to the selectivity observed with (+)-26. Although the DNA alkylation selectivity of (+)-62 and (+)-64 potentially could have been significantly altered or have become increasingly more tolerant of a GC base-pair in the alkylation sequence, the selectivity proved more revealing than this simple expectation. Not only did (+)-62 and (+)-64 alkylate DNA with the near identical selectivity of (+)-26, but the unnatural enantiomer selectivity for 62 and 64 proved essentially identical to that of ent-(xe2x88x92)-26. Thus, in a manner essentially identical to (+)- and ent-(xe2x88x92)-26 which exhibit distinct alkylation selectivities (5xe2x80x2-A/TA/TA/TA versus 5xe2x80x2-A/TAA/TA/T, respectively) characteristic of the reverse binding orientations and offset 3.5 base-pair AT-rich binding sites surrounding the alkylation site, the two entantiomers of 62 and 64 alkylated essentially the same sites as the corresponding enantiomers of 26 within duplex DNA. Moreover, this was observed to occur not with the increasing tolerance for incorporation of GC base-pairs in the alkylation sequence, but rather with a diminished DNA alkylation efficiency (100xc3x97) relative to that of (+)- and ent-(xe2x88x92)-CBI-CDPI1 (26). The potential origin of these effects have been discussed elsewhere.
The cytotoxic properties of 62-65 and that of the closely related CBI agents are summarized in FIG. 13. Consistent with their relative efficiencies of DNA alkylation, the natural enantiomers of 62 and 64 were essentially indistinguishable (500-1000 pM, L1210) and 100-200xc3x97 less potent than (+)-CBI-CDPI1 (26). Thus, the introduction of the single nitrogen atom in the DNA binding subunit of 64 reduced the biological potency 100 to 200-fold. Consistent with prior observations, the natural enantiomers of 62 and 64 were 10-100xc3x97 more potent than the corresponding unnatural enantiomers. CBI-Indole-NMe3+: Electropositive Substituents Capable of Enhancing DNA
Alkylation Efficiency Through Stabilizing Electrostatic Interactions. In recent studies, we have studied the impact that electronegative and electropositive substituents placed on peripheral face of the agents have on the noncovalent DNA binding affinity and selectivity. In these studies, we defined a destabilizing contribution to the DNA binding affinity that results from the introduction of a strong electronegative substituent and described a substantial enhancement of noncovalent binding affinity that results from introduction of an electropositive substituent. This was attributed to a spatially well-defined destabilizing or stabilizing electrostatic interaction with the negatively charged DNA phosphate backbone, respectively, and was found to have little impact on the intrinsic AT-rich binding selectivity of the parent agents. These studies were recently extended to the preparation of 66-68, close analogs of 28/33, containing a peripheral quaternary ammonium salt capable of providing a strong, stabilizing electrostatic interaction with the DNA phosphate backbone. Consistent with expectations, the agents 66-68 alkylated DNA with the same relative efficiency as 1-2 and were approximately 100xc3x97 more effective than 28 or 33 which lack the ammonium salt substituent. Because of the smaller size of the agents, they exhibited a DNA alkylation selectivity that was subtly altered from that of (+)-CC-1065, but comparable to that of (+)-duocarmycin SA. In addition, the agents were water soluble and offer potential advantages over the existing agents.
Consequently, we were interested in the relative cytotoxic properties of 66-68 and the results of their evaluations are summarized in FIG. 15. Although 66-68 were essentially identical in their cytotoxic potencies (10 nM), they proved to be slightly less potent than (+)-CBI-indole1 (28) and approximately 1000xc3x97 less potent than (+)-1 and (+)-2. This is in contrast to expectations based on their relative efficiencies of DNA alkylation. Although this was not investigated, we attribute this diminished cytotoxic potency to ineffective cellular penetration required for the agents to reach their intracellular target.
Additional Analogs. In the course of our investigations, several additional agents have been examined including 73 and 75, simple derivatives of the CBI alkylation subunit which possess enhanced DNA alkylation capabilities and in vitro cytotoxic potency by virtue of stabilizing electrostatic DNA binding. That is, in place of the DNA binding affinity derived from hydrophobic binding and stabilizing van der Waals contacts provided by the central and right-hand subunits of 1-3, the simple electrostatic binding affinity provided by the protonated amine of 73 and 75 with the negatively charged phosphate backbone of DNA proved sufficient to substantially enhance the DNA alkylation intensity and in vitro cytotoxic activity.
The semicarbazide of CBI and its seco chloride precursor were prepared as detailed in FIG. 16. Treatment of bis(2,4-dinitrophenyl)carbonate (69) with tert-butylcarbazate (70, 1 equiv, 24xc2x0 C., 2 h, EtOAc) provided 71 (61%) and a convenient acylating agent for introduction of the tert-butyloxycarbonyl protected hydrazide. N-deprotection of 15 (3 N HCl-EtOAc, 24xc2x0 C., 20 min, 100%) followed by immediate treatment of the unstable amine hydrochloride salt 16 with 71 (1.3 equiv, 1 equiv Et3N, 24xc2x0 C., 5.5 h, THF, 91%) provided 72 in excellent yield. Acid-catalyzed N-BOC deprotection of 72 provided 73 and exposure of 72 or 73 to 5% aqueous NaHCO3-THF (24xc2x0 C.) provided 74 or 75, respectively.
The results of the in vitro cytotoxic evaluation of the N-semicarbazide of CBI conducted on its more stable seco precursor 73 are detailed in FIG. 18 along with the comparative results from the evaluation of N-BOC-CBI (15) and 72. Notably, 73 which possesses the free amine exhibited more potent in vitro cytotoxic activity than its precursor possessing the tert-butylcarbazate (72, ca. 100xc3x97) or N-BOC-CBI (9) itself, and proved to be only 100xc3x97 less potent than (+)-CC-1065.
Consistent with the trends observed in the relative cytotoxic potency of the agents, the intensity of DNA alkylation similarly increased with the introduction of the free semicarbazide and the results of these studies have been detailed elsewhere. Thus, the introduction of a positively charged functionality (protonated amine) onto the simple CBI alkylation subunit served to enhance the DNA alkylation intensity of the agent presumably by providing noncovalent electrostatic DNA binding affinity to the agents. Consistent with the enhancement in the DNA alkylation intensity (100xc3x97), the in vitro cytotoxic activity of the agents increased correspondingly (100xc3x97).
The introduction of a terminal semicarbazide onto CBI-CDPI2 was carried for comparison purposes (FIG. 17). Acid-catalyzed deprotection of N-BOC-CDPI2 (76, CF3CO2H, 25xc2x0 C., 1 h) followed by coupling of crude amine salt with 71 (1.5 equiv, 1 equiv Et3N, 25xc2x0 C., 19 h, 91% overall) provided 77 in excellent conversion. Direct coupling of 77 with freshly generated 16 (3 equiv EDCI, DMF, 25xc2x0 C., 10 h) provided 78 (65%) in good conversions. Acid-catalyzed deprotection (3M HCl-EtOAc, 25xc2x0 C., 30 min) cleanly provided 79 (95-100%).
The examination of 78 and 79 revealed that this alteration in the C-terminus of CBI-CDPI2 (25) did not impact on the inherent properties of the agent, FIG. 18. Thus, in contrast to 73 where the introduction of a stabilizing electrostatic interaction enhances the DNA alkylation efficiency and cytotoxic potency of the agent, it had no impact on the properties of 79 versus 78/25. Presumably, this may be attributed to the fact that the noncovalent hydrophobic binding affinity of 25 is already sufficient to provide fuill stabilization of the reversible DNA adduct and the maximal cytotoxic potency and that the additional electrostatic stabilization provided in 79 is unnecessary.
Notably, the terminal acyl hydrazides of 73, 75 and 79 may serve as useful functionality for subsequent reversible or irreversible conjugation with tumor selective delivery systems and such studies are underway.
In contrast to early speculation, deep-seated modifications in the CC-1065 and duocarmycin alkylation subunit are well tolerated and the CBI-based analogs proved to be potent cytotoxic agents and efficacious antitumor compounds. A direct relationship between functional stability and cytotoxic potency was defined and validated. As such, the readily accessible CBI-based analogs were found to be 4xc3x97 more stable and 4xc3x97 more potent than the corresponding analogs containing the CPI alkylation subunit of CC-1065 and comparable in potency to the agents containing the duocarmycin SA alkylation subunit. Similarly, the CBI-based agents alkylate DNA with an unaltered sequence selectivity at an enhanced rate and with a greater efficiency than the corresponding CPI analogs and were comparable to the corresponding DSA analog. Systematic modification and simplification of the attached DNA binding subunits have provided a series of synthetic and potent cytotoxic agents including 25-29 and 57-61 whose biological profile are under further study. A number of the agents detailed herein exhibit potent and efficacious antitumor activity.
One aspect of the invention is directed to a compound represented by the following structure: 
wherein R1 is selected from the group consisting of xe2x80x94CH2CH3 (alkyl), xe2x80x94NHCH3 (-N-alkyl), xe2x80x94OCH3 (O-alkyl), xe2x80x94NH2, xe2x80x94NHNH2, xe2x80x94NHNHCO2tBu, and a radical. The radical is represented by the following structure: 
wherein A is selected from the group consisting of NH and O; B is selected from the group consisting of C and N; R2 is selected from the group consisting of hydrogen, hydroxyl, O-alkyl (C1-C6), N-alkyl (C1-C6)3 and a first N-substituted pyrrolidine ring; R3 is selected from the group consisting of hydrogen, hydroxyl, O-alkyl (C1-C6), N-alkyl (C1-C6)31 the first N-substituted pyrrolidine ring; R4 is selected from the group consisting of hydrogen, hydroxyl, O-alkyl (C1-C6), and N-alkyl (C1-C6)3; R5 is selected from the group consisting of hydrogen, hydroxyl, O-alkyl (C1-C6), and N-alkyl (C1-C6)3; and V1 represents a first vinylene group between R2 and R3. The following provisos apply: if R2 participates in the first N-substituted pyrrolidine ring, then R3 also particlates in the first N-substituted pyrrolidine ring; if R3 participates in the first N-substituted pyrrolidine ring, then R2 also particlates in the first N-substituted pyrrolidine ring; if R2 and R3 participate in the first N-substituted pyrrolidine ring, then R4 and R5 are hydrogen; and if R2 is hydrogen, then R4 and R5 are hydrogen and R3is N-alkyl (C1-C6)3. The first N-substituted pyrrolidine ring is fused to the first vinylene group between R2 and R3 and is represented by the following structure: 
wherein V1 represents the first vinylene group between R2 and R3; R6 is selected from the group consisting of xe2x80x94CH2CH3 (alkyl), xe2x80x94NHCH3 (-N-alkyl), xe2x80x94OCH3 (O-alkyl), xe2x80x94NH2, xe2x80x94NHNH2, xe2x80x94NHNHCO2tBu, and a radical. The radical is represented by the following structure: 
wherein C is selected from the group consisting of NH and O; D is selected from the group consisting of C and N; R7 is selected from the group consisting of hydrogen, hydroxyl, O-alkyl (C1-C6), N-alkyl (C1-C6)3, and a second N-substituted pyrrolidine ring; R8 is selected from the group consisting of hydrogen, hydroxyl, O-alkyl (C1-C6), N-alkyl (C1-C6)3, the second N-substituted pyrrolidine ring; and V2represents the second vinylene group between R7 and R8. The following provisos apply: if R7 participates in the N-substituted pyrrolidine ring, then R8 also particlates in the N-substituted pyrrolidine ring; and if R8 participates in the N-substituted pyrrolidine ring only if R7 also particlates in the N-substituted pyrrolidine ring. The second N-substituted pyrrolidine ring is fused to the second vinylene group between R7 and R8 and is represented by the following structure: 
wherein V2 represents the second vinylene group between R7 and R8; and R9 is selected from the group consisting of xe2x80x94CH2CH3 (alkyl), xe2x80x94NHCH3 (-N-alkyl), xe2x80x94OCH3 (O-alkyl), xe2x80x94NH2, xe2x80x94NHNH2, and xe2x80x94NHNHCO2tBu.
Another aspect of the invention is directed to a compound represented by the following structure: 
wherein X is selected from the group consisting of chlorine, bromine, iodine, and OTOS; and R1 is selected from the group consisting of xe2x80x94CH2CH3 (alkyl), xe2x80x94NHCH3 (-N-alkyl), xe2x80x94OCH3 (O-alkyl), xe2x80x94NH2, xe2x80x94NHNH2, xe2x80x94NHNHCO2tBu, and a radical. The radical is represented by the following structure: 
wherein A is selected from the group consisting of NH and O; B is selected from the group consisting of C and N; R2 is selected from the group consisting of hydrogen, hydroxyl, O-alkyl (C1-C6), N-alkyl (C1-C6)3 and a first N-substituted pyrrolidine ring; R3 is selected from the group consisting of hydrogen, hydroxyl, O-alkyl (C1-C6), N-alkyl (C1-C6), the first N-substituted pyrrolidine ring; R4 is selected from the group consisting of hydrogen, hydroxyl, O-alkyl (C1-C6), and N-alkyl (C1-C6)3; R5 is selected from the group consisting of hydrogen, hydroxyl, O-alkyl (C1-C6), and N-alkyl (C1-C6)3; and V1 represents a first vinylene group between R2 and R3. The following provisos apply: if R2 participates in the first N-substituted pyrrolidine ring, then R3 also particlates in the first N-substituted pyrrolidine ring; if R3 participates in the first N-substituted pyrrolidine ring, then R2 also particlates in the first N-substituted pyrrolidine ring; if R2 and R3 participate in the first N-substituted pyrrolidine ring, then R4 and R5 are hydrogen; and if R2 is hydrogen, then R4 and R5 are hydrogen and R3 is N-alkyl (C1-C6)3. The first N-substituted pyrrolidine ring is fused to the first vinylene group between R2 and R3 and is represented by the following structure: 
wherein V1 represents the first vinylene group between R2 and R3; R6 is selected from the group consisting of xe2x80x94CH2CH3 (alkyl), xe2x80x94NHCH3 (-N-alkyl), xe2x80x94OCH3 (O-alkyl), xe2x80x94NH2, xe2x80x94NHNH2, xe2x80x94NHNHCO2tBu, and a radical. The radical is represented by the following structure: 
wherein C is selected from the group consisting of NH and O; D is selected from the group consisting of C and N; R7 is selected from the group consisting of hydrogen, hydroxyl, O-alkyl (C1-C6), N-alkyl (C1-C6)3, and a second N-substituted pyrrolidine ring; R8 is selected from the group consisting of hydrogen, hydroxyl, O-alkyl (C1-C6), N-alkyl (C1-C6)3, the second N-substituted pyrrolidine ring; and V2represents the second vinylene group between R7 and R8. The following provisos apply: if R7 participates in the N-substituted pyrrolidine ring, then R8 also particlates in the N-substituted pyrrolidine ring; and if R8 participates in the N-substituted pyrrolidine ring only if R7 also particlates in the N-substituted pyrrolidine ring. The second N-substituted pyrrolidine ring is fused to the second vinylene group between R7 and R8 and is represented by the following structure: 
wherein V2 represents the second vinylene group between R7 and R8; and R9 is selected from the group consisting of xe2x80x94CH2CH3 (alkyl), xe2x80x94NHCH3 (-N-alkyl), xe2x80x94OCH3 (O-alkyl), xe2x80x94NH2, xe2x80x94NHNH2, and xe2x80x94NHNHCO2tBu.
Another aspect of the invention is directed to a compound represented by the following structure: 
wherein A is selected from the group consisting of NH and O and B is selected from the group consisting of NH, O, and S.
Another aspect of the invention is directed to a compound compound represented by the following structure: 
Another aspect of the invention is directed to a compound compound represented by the following structures: 
Another aspect of the invention is directed to a compound compound represented by the following structure: 
Another aspect of the invention is directed to a compound compound represented by the following structure: 
Another aspect of the invention is directed to a compound compound represented by the following structure: 
where R is selected from the group comprising of: H, 5-NMe3+, 6-NMe3+, 7-NMe3+.
Another aspect of the invention is directed to a compound compound represented by the following structure: 
where R is selected from the group comprising of: CO2tBu, Hxe2x80x94HCl.
Another aspect of the invention is directed to a compound compound represented by the following structure: 
Another aspect of the invention is directed to a compound compound represented by the following structure: 
Another aspect of the invention is directed to a compound compound represented by the following structure: 
Another aspect of the invention is directed to a compound compound represented by the following structure: 
Another aspect of the invention is directed to a compound compound represented by the following structure: 
where R is selected from the group comprising of: Hxe2x80x94HCl, CONHMe, CO2CH3, COEt.
Another aspect of the invention is directed to a compound compound represented by the following structure: 
Another aspect of the invention is directed to a compound compound represented by the following structure: 
wherein A is selected from the group consisting of O and R is selected from the group consisting of NO2 and NH2.
Another aspect of the invention is directed to a compound compound represented by the following structure: 
wherein B is selected from the group consisting of O and S.
Another aspect of the invention is directed to a compound compound represented by the following structure: 
wherein A is selected from the group consisting of NH and O and B is selected from the group consisting of NH, O, and S and R is selected from the group consisting of H and CH3.
Another aspect of the invention is directed to a compound compound represented by the following structure: 
wherein A is selected from the group consisting of NH and O and B is selected from the group consisting of NH, O, and S.